CN106048064B - SSR molecular marker for identifying female sex of pistacia chinensis bunge and primer pair thereof - Google Patents
SSR molecular marker for identifying female sex of pistacia chinensis bunge and primer pair thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular genetic breeding, in particular to SSR molecular markers and primer pairs thereof for identifying the female sex of pistacia chinensis bunge.
Description
Technical Field
The invention relates to the technical field of molecular genetic breeding, in particular to SSR molecular markers for identifying female sex of pistacia chinensis bunge and primer pairs thereof.
Background
Pistacia chinensis Bunge (Pistacia chinensis Bunge) is a Pistacia chinensis Bunge of Anacardiaceae, a heterotrophic deciduous arbor plant of types, and is a very important potential biomass energy tree species, the oil content of the seed of Pistacia chinensis Bunge is as high as 40%, and is nondrying oil, the cetane number of the prepared biodiesel is as high as 51.3, and the prepared biodiesel meets the European Union standard with the highest requirement.
At present, in the process of industrialized development of pistacia chinensis bunge, the construction and development of a raw material base are slow due to insufficient supply and demand of raw materials of pistacia chinensis bunge, the industrialized development of the pistacia chinensis bunge is severely restricted by the shortage problem of the raw materials of the pistacia chinensis bunge, meanwhile, the pistacia chinensis bunge is taken as a biomass energy plant, the seeds of the pistacia chinensis bunge are required to be used for preparing the biodiesel, the number of female plants is expected to be far greater than that of male plants because the pistacia chinensis bunge is a male plant of a heterofemale plant and can only provide pollen, in addition, the pistacia chinensis bunge is difficult to distinguish the male plants and the female plants in the childhood, the childhood period is as long as more than 5-8 years, the genome information of the.
Therefore, a large amount of SSRs are developed by utilizing a second-generation sequencing technology, the SSR molecular markers which are closely linked with the pistacia chinensis bunge gender are found by combining a BSA (bovine serum albumin) method, a molecular marker auxiliary selection system suitable for high-throughput, rapid and accurate identification of the early-stage gender of the pistacia chinensis bunge is established, molecular evidences can be provided for a pistacia chinensis bunge gender decision mechanism, the pistacia chinensis bunge molecular selection breeding process is promoted, and a reliable early-stage gender identification technology can be provided for industrialized production of the pistacia chinensis bunge.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The th purpose of the invention is to provide SSR molecular marker PCSR 55 primer pairs, which have good specificity and high amplification efficiency and can be used for sex identification of pistacia chinensis bunge.
The second purpose of the invention is to provide methods for identifying the sex of pistacia chinensis bunge, which can solve the problems of long time and low efficiency in identifying the sex of the pistacia chinensis bunge in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the SSR molecular marker PCSSR55 primer pair has the nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2.
In the development history of such hermaphrodite plants as pistacia chinensis bunge, it is difficult to distinguish the male plants from the female plants in the juvenile period. The DNA molecular marker is not influenced by the development time and the group length specificity, and the early sex identification of the hermaphrodite plants can obtain reliable identification information which cannot be obtained by a common method. And has the advantages of rapidness, high flux and accuracy.
The invention obtains SSR in batches by carrying out transcriptome sequencing on female and male flower buds of pistacia chinensis bunge, designs primers, autonomously constructs gene pools of female and male plants, screens the gene pools by using autonomously developed SSR molecular markers, carries out group verification, and finally obtains the SSR molecular marker PCSSR55 which can be used for early sex identification of the pistacia chinensis bunge, thereby forming the technical scheme of the invention.
Generally, the primer specificity is increased 4-fold for each nucleotides, so that the shortest primer length for most applications is 18 nucleotides.
the length of the primer is 18-30 bases, the length of the primer is 22 and 23 bases, the primer is relatively short, due to entropy, the rate of annealing and combining the short primer to the target DNA to form a stable double-stranded template for DNA polymerase to combine is higher, and therefore the primer has high amplification efficiency.
the G + C content in the primer sequence is 40% -60%, the GC content and Tm value of pair of primers should be coordinated.
The primer has no complementary sequence (continuous complementary base is less than 3 bp); the 3' end of the primer is not modified and is not easy to mismatch; the length of the final amplification product is 151bp, and the target fragment is easy to successfully amplify.
In conclusion, the primer has good specificity and high amplification efficiency, and can be well used for molecular identification of female sex of pistacia chinensis bunge.
SSR molecular markers PCSSR55, and the nucleotide sequence of the molecular markers is shown as SEQ ID NO. 3.
Because the flanking sequences of a specific SSR are usually single sequences with strong conservation, primers can be synthesized according to the flanking sequences for PCR amplification, so that a single microsatellite locus is amplified.
The primer pair and the SSR molecular marker are applied to sex identification of pistacia chinensis bunge.
The primer pair and the SSR molecular marker are applied to the auxiliary breeding related to the sex of the pistacia chinensis bunge.
method for identifying the sex of Pistacia chinensis Bunge, comprising the following steps:
amplifying the genome DNA of the pistacia chinensis bunge to be detected by utilizing the primer pair;
and carrying out electrophoretic identification on the amplification product, wherein if the product contains a strip which is consistent with the molecular weight of the molecular marker, the pistacia chinensis bunge to be detected is a female plant, and otherwise, the pistacia chinensis bunge is a male plant.
Preferably, in the method for identifying the sex of pistacia chinensis bunge, in an amplification reaction system, the concentration of the upstream primer and the downstream primer is 0.1-0.3 mu M, and the concentration of the DNA template is more than or equal to 4 ng/mu l.
The invention relates to amounts of components in system or ratio, which should be regarded as the limit of proportional relation between related components, but not the absolute value of each dosage, and the proportional relation between components can be changed properly.
Taq DNA polymerase and Mg are also needed for amplification2+、dNTPs and buffer solution. Taq DNA polymerase has good thermal stability and can still maintain higher catalytic activity under the high-temperature condition of PCR circulation; mg (magnesium)2+Is coenzyme of Taq DNA polymerase, and the concentration of the coenzyme directly influences the activity and specificity of the enzyme; dNTPs are raw materials for DNA amplification; the buffer can provide suitable catalytic conditions for amplification of Taq DNA polymerase.
Preferably, the method for identifying the sex of pistacia chinensis bunge as described above, wherein the reaction procedure of amplification is as follows:
the annealing temperature for PCR amplification is usually 40 ℃ to 60 ℃. The annealing temperature is determined by the Tm value of the primer, and within the allowable range of the Tm value, the non-specific binding between the primer and the template can be greatly reduced by selecting higher annealing temperature, so that the specificity of the PCR reaction is improved. The primer adopted by the invention has the annealing temperature of 56 ℃ and higher specificity.
As the reaction proceeds, the enzyme is gradually inactivated, the dNTPs and other raw materials are gradually consumed, and the amplification of non-specific products is correspondingly increased, so that the number of reaction cycles is increased, but the number of cycles is still not too large, and the invention is limited to 38-42 cycles.
Preferably, in the method for identifying the sex of pistacia chinensis bunge as described above, the gel used in the electrophoretic identification is 5-7% polyacrylamide gel.
The method for obtaining the SSR molecular marker comprises the following steps:
1) performing transcriptome sequencing on the single flower buds of the female and the single plant of the Liangmu realgar respectively, analyzing SSR distribution characteristics in sequencing data and designing SSR primers;
2) constructing a female realgar and juniper gene pool, and screening the gene pool by using the SSR primer;
3) and carrying out sex verification by using the group for constructing the gene pool.
Preferably, in the method for obtaining the SSR molecular marker, in step 2), the sample size used for constructing the female and realgar ceruleus wood gene pool is as follows:
18-25 female plants and 18-25 male plants.
Compared with the prior art, the invention has the beneficial effects that:
1) the method can identify the sex of pistacia chinensis bunge at the early growth stage of the pistacia chinensis bunge, avoids the waste time and economic cost consumption, and has the accuracy rate of identifying the sex of the pistacia chinensis bunge of more than 95 percent, rapidness, simplicity and practicability.
2) The primer used in the invention has good specificity and high amplification efficiency, and the PCR reaction can be completed by less template amount, so that the primer can be well used for molecular identification of the sex of pistacia chinensis bunge.
3) Many molecular identification experiments require two or more pairs of primers to amplify a plurality of amplification products, some methods also require enzyme digestion reaction to carry out auxiliary identification, the operation undoubtedly increases the time cost and the money cost of identification, the pairs of primers also easily cause cross contamination among the primers, different primers can also be subjected to non-specific combination, meanwhile, Tm values among different primers can also have differences and influence the selection of annealing temperature, and further influence the amplification efficiency.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a polyacrylamide gel electrophoresis diagram of the molecular marker PCSSR55 amplified in gene pool and population screening in the embodiment of the invention;
FIG. 2 is a polyacrylamide gel electrophoresis image of the molecular marker PCSSR55 amplified in the female and male strains of the Pistacia chinensis Bunge natural population in the embodiment of the invention.
English-Chinese translation
1. SSR (simple Sequence repeats) marker is molecular marker technologies based on specific primer PCR developed in recent years, also called microsatellite DNA (Microsatelite DNA), and is series repetitive sequences which are composed of several nucleotides (1-6 in ) as repetitive units and are dozens of nucleotides in length.
2. And (3) PCR: polymerase Chain Reaction, is an in vitro nucleic acid amplification technology developed in the middle of the 80 s.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Obtaining of molecular marker PCSSR 55.
1) Performing transcriptome sequencing on the flower buds of the single female realgar and the single realgar lianmu plant respectively;
materials: in 2016, 1 each of male and female plants was selected in 1 month in southwest forestry university campus of Kunming, Yunnan, and female and male flower buds were collected and stored in liquid nitrogen. Samples were sent to Hangzhou and one King technologies, Inc., which were entrusted with completing transcriptome sequencing work.
2) Analyzing sequencing data, excavating SSR, analyzing SSR distribution characteristics and designing SSR primers;
search of transcriptome microsatellites using the local search software Perl script MicroSatellite (MISA). SSR locus search motif is 2 to 6 nucleotide repeats, and the search criteria are as follows: 2-, 3-, 4-, 5-, 6-, and 7-. nucleotide repeats at least greater than 5, 4, 3, 2, and 2 times, respectively. SSR loci obtained by MISA search were provided with forward and reverse primers on both sides, respectively, and Primer 3 was used for Primer design. Primer 3 design primers the following criteria were used: the length of the positive and negative direction primers is 18bp-28bp, and 23bp is most suitable; the Tm temperature of the primer is 55-65 ℃, and 60 ℃ is most suitable; the size of the amplified fragment is expected to be 80bp-160 bp.
3) Constructing a female gene pool and a male gene pool (20 female plants and 20 male plants), and screening the gene pools by SSR primers;
the DNA templates of the constructed female and male gene pools (20 female strains and 20 male strains) are amplified by utilizing the synthesized SSR, and SSR primers which express polymorphism between the female and male gene pools are screened.
4) And carrying out sex verification by using the group for constructing the gene pool.
DNA templates of 20 female strains and 20 male strains are amplified by SSR markers which show polymorphism between female and male gene pools, the molecular marker PCSSR55 obtained by screening can be amplified in the female strains to obtain two bands, only the 19 th strain in the male strains is amplified to obtain two bands, and the other male strains are bands.
The amplification results are shown in FIG. 1.
Example 2
Application of molecular marker PCSSR 55R.
Collecting tender leaves of 100 parts of female plants and 102 parts of male plants in a natural population of Chenjiagou Pistacia chinensis Bunge of Anyang city, Henan province, China, and extracting plant genome DNA; 202 pistacia chinensis bunge DNA samples were amplified by using molecular marker primers PCSSR55F and PCSSR 55R.
The amplified reaction system comprises the following components in terms of 10 μ l of reaction system:
the PCR amplification procedure was:
FIG. 2 shows the polypropylene gel electrophoresis image of the present molecular marker PCSSR55 amplified in the female and male strains of the Pistacia chinensis Natural population:
the invention amplifies the samples of 100 parts of female plants and 102 parts of male plants, two spectral bands are amplified in 100 parts of female plants, and the size of the specific spectral band fragment is 151 bp;
of all 102 males, only 4 amplified two bands, and the remaining 98 males all had amplified bands.
In recent years, China speeds up the pace of developing the biodiesel industry, and places all directions focus on several fuel oil trees with more perfect production technology, wherein pistacia chinensis bunge is the focus of biodiesel raw materials in recent years with the continuous development and utilization of pistacia chinensis bunge resources, the application range of the pistacia chinensis bunge is continuously expanded, the market demand is greatly increased, a large number of oil-containing seeds are reasonably planted and obtained in a short time, and the problem is the current primary solution.
According to the method, the sex identification can be carried out in the early growth stage of the pistacia chinensis bunge, the useless time cost and economic cost consumption are avoided, and according to the record in the embodiment 2, the accuracy rate of the method for identifying the sex of the pistacia chinensis bunge can reach more than 95%, and the method is rapid, simple and feasible.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1, application of primer pairs of SSR molecular marker PCSSR55 and SSR molecular marker PCSSR55 in identifying the sex of pistacia chinensis bunge;
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 3.
2, application of primer pairs of SSR molecular marker PCSSR55 and SSR molecular marker PCSSR55 in auxiliary breeding related to the sex of pistacia chinensis bunge;
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the nucleotide sequence of the molecular marker is shown as SEQID NO. 3.
3, method for identifying the sex of Pistacia chinensis Bunge, which is characterized by comprising the following steps:
amplifying the genome DNA of the pistacia chinensis bunge to be detected by using the primer pair of the PCSSR55 of the SSR molecular marker;
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 3;
and (3) carrying out electrophoretic identification on the amplification product, wherein if the product contains a band which is consistent with the molecular weight of the SSR molecular marker PCSSR55, the pistacia chinensis bunge to be detected is a female plant, and otherwise, the pistacia chinensis bunge is a male plant.
4. The method for identifying the sex of pistacia chinensis bunge according to claim 3, wherein in an amplification reaction system, the concentration of an upstream primer and a downstream primer is 0.1-0.3 mu M, and the concentration of a DNA template is more than or equal to 4 ng/mu l.
5. The method for sexing pistacia chinensis bunge according to claim 3, wherein the reaction procedure of amplification is as follows:
①94℃ 2~4min；
②94℃ 25~35s；
③56℃ 25~35s；
④72℃ 0.8~1.2min；
⑤ repeating ②③④ cycles for 38-42 cycles;
⑥72℃ 4~6min。
6. the method for sexing pistacia chinensis bunge according to claim 3, wherein the gel used in the electrophoretic identification is 5% -7% polyacrylamide gel.
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